WO2023072145A1 - 一种纯化单链dna的方法 - Google Patents

一种纯化单链dna的方法 Download PDF

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WO2023072145A1
WO2023072145A1 PCT/CN2022/127682 CN2022127682W WO2023072145A1 WO 2023072145 A1 WO2023072145 A1 WO 2023072145A1 CN 2022127682 W CN2022127682 W CN 2022127682W WO 2023072145 A1 WO2023072145 A1 WO 2023072145A1
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stranded dna
binding protein
product
purification
purifying
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陈娟
叶露萌
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南京金斯瑞生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present application relates to the field of biotechnology, in particular to a method for purifying single-stranded DNA.
  • CRISPR/Cas9 technology is a technology in which single-stranded gRNA (Guide RNA)-mediated Cas9 nuclease performs directional modification on the DNA sequence of the target gene, which can efficiently realize specific gene knockout, knock-in and site-specific modification functions.
  • Single-stranded DNA as a template for HDR Homology directed repair, homology-mediated double-stranded DNA repair
  • CRISPR/Cas9 gene knock-in Knock-in
  • the single-stranded DNA template is very suitable for the insertion, replacement and correction of genes based on CRISPR technology, and is an ideal technical solution for gene editing of primary cells, stem cells, and establishment of genetically modified animal models.
  • WO2020135651 discloses the automatic circularization method of double-stranded DNA mediated by uracil-specific subtractive enzyme (USER) and type II restriction endonuclease precise cutting, isothermal
  • the rolling circle replication (RCA) of amplifying single-stranded DNA is cleverly combined to prepare single-stranded DNA samples.
  • This method also increases the preparation yield of single-stranded DNA samples from conventional several ug to tens of ug.
  • DISR uracil-specific subtractive enzyme
  • RCA rolling circle replication
  • One of the embodiments according to the specification provides a method for purifying single-stranded DNA.
  • the method includes: mixing a sample containing single-stranded DNA with a single-stranded DNA binding protein and incubating for a first time to obtain a first product, which contains the single-stranded DNA and the single-stranded DNA binding protein The first product is purified to obtain a target product containing purified single-stranded DNA.
  • the final concentration of the single-stranded DNA-binding protein is 0.5 ng/ ⁇ L-500 ng/ ⁇ L.
  • the final concentration of the single-stranded DNA-binding protein is 1 ng/ ⁇ L-200 ng/ ⁇ L.
  • the first time is 30 minutes to 12 hours.
  • the first time is 2-4 hours.
  • the sample is mixed with the single-stranded DNA binding protein and incubated at 30-37°C.
  • the step of purifying the first product includes: separating and purifying the first product to obtain a second product; purifying the second product to at least remove the single-stranded DNA binding protein to obtain the target product containing purified single-stranded DNA.
  • the method for separating and purifying the first product is affinity chromatography, ion exchange chromatography or gel filtration chromatography.
  • the method for separating and purifying the first product is affinity chromatography.
  • the single-stranded DNA binding protein is tagged for purification.
  • the purification tag is a polyhistidine tag, a glutathione-S transferase tag, a FLAG tag or a Strep-tagII tag.
  • the method for purifying the second product is selected from at least one of the following methods: phenol-chloroform extraction method, DNA adsorption spin column recovery method, plasmid extraction ion exchange column recovery method, Isopropanol precipitation, ethanol precipitation, and molecular sieve chromatography.
  • the method further includes performing PCR amplification and DNA sequencing on the target product to verify whether the target product contains the complete sequence of the single-stranded DNA.
  • the single-stranded DNA binding protein comprises Escherichia coli, phi29 phage, Salmonella phage Vi II-E1, thermophile HB8, Klebsiella Phi K02 phage, Yersinia Py54 phage Or the single-stranded DNA binding protein of Synechococcus bacteriophage S-CBS2, its core functional fragment, its homolog sequence or its mutant sequence.
  • the single-stranded DNA binding protein comprises a sequence with at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 9, 10 or 11 and has the function of binding to single-stranded DNA.
  • One of the embodiments of the present specification provides a kit for purifying single-stranded DNA, including a single-stranded DNA binding protein and a purification reagent.
  • the purification reagent is an affinity chromatography purification reagent.
  • One of the embodiments of the present specification provides the use of single-stranded DNA binding protein in purifying single-stranded DNA.
  • Figure 1 is a schematic diagram of the structure of a recombinant expression vector according to some embodiments of the present description
  • Fig. 2 is the SDS-PAGE and Western Blot analysis figure of the single-stranded DNA binding protein under reducing conditions according to some embodiments of the specification;
  • Fig. 3 is a comparison of agarose gel electrophoresis diagrams before and after purification of the target sequence 1 single-stranded DNA according to some embodiments of the specification;
  • Fig. 4 is a schematic diagram of Sanger sequencing verification results of the target sequence 1 shown according to some embodiments of the specification;
  • Fig. 5 is a comparison of agarose gel electrophoresis diagrams before and after purification of the target sequence 2 single-stranded DNA according to some embodiments of the specification;
  • Fig. 6 is a schematic diagram of Sanger sequencing verification results of target sequence 2 according to some embodiments of the present specification.
  • Single-stranded DNA binding protein (Single-Stranded DNA binding protein, SSB) is a kind of protein that can bind to single-stranded DNA. The strand portion is not degraded by nucleases. This specification proposes that single-stranded DNA can be purified on a large scale and efficiently by utilizing the binding ability of single-stranded DNA-binding proteins to single-stranded DNA, and higher yields can be obtained.
  • a method of purifying single-stranded DNA is provided.
  • the method includes mixing a sample containing single-stranded DNA with a single-stranded DNA binding protein and incubating for a first time to obtain a first product comprising the single-stranded DNA and the A combination of single-stranded DNA binding proteins; the first product is purified to obtain a target product containing purified single-stranded DNA.
  • the sample may be a product obtained from a large-scale production process of single-stranded DNA, so the sample may contain non-specific amplification products, such as double-stranded DNA.
  • the traditional method is to use gel electrophoresis to recover single-stranded DNA in the sample, but the yield of this method is not high, and it greatly increases the time and labor costs of the downstream purification process, hindering large-scale preparation process.
  • the single-stranded DNA-binding protein that binds to single-stranded DNA can be purified on a large scale and efficiently by utilizing the single-stranded DNA-binding ability of the single-stranded DNA-binding protein.
  • the sample may refer to any sample containing single-stranded DNA at a certain purity.
  • the proportion of single-stranded DNA in the sample may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc., which is not limited in this specification.
  • the single-stranded DNA is also called the target sequence.
  • the final concentration of the single-stranded DNA-binding protein when the sample is mixed with the single-stranded DNA-binding protein, the final concentration of the single-stranded DNA-binding protein may be 0.1 ng/ ⁇ L-1000 ng/ ⁇ L. In some embodiments, when the sample is mixed with the single-stranded DNA-binding protein, the final concentration of the single-stranded DNA-binding protein may be 0.5 ng/ ⁇ L-500 ng/ ⁇ L. In some embodiments, when the sample is mixed with the single-stranded DNA-binding protein, the final concentration of the single-stranded DNA-binding protein may be 1 ng/ ⁇ L-200 ng/ ⁇ L.
  • the first time of incubating the sample after mixing with the single-stranded DNA binding protein may be 20 minutes to 24 hours. In some embodiments, the first time of incubating the sample after mixing with the single-stranded DNA binding protein may be 30 minutes to 15 hours. In some embodiments, the first time for incubating the sample after mixing with the single-stranded DNA-binding protein can be 30 minutes to 12 hours, for example, it can be 2 hours or 4 hours. In some embodiments, the first time of incubating the sample after mixing with the single-stranded DNA binding protein may be 30 minutes to 6 hours. In some embodiments, the first time of incubating the sample after mixing with the single-stranded DNA binding protein may be 2-4 hours.
  • the sample is mixed with the single-stranded DNA binding protein and incubated at 30-37°C.
  • the temperature of the incubation may be 30°C, 32°C, 35°C, 37°C, etc.
  • the step of purifying the first product may include separating and purifying the first product to obtain a second product; purifying the second product to at least remove the stranded DNA binding protein to obtain the target product containing purified single-stranded DNA.
  • the first product is separated and purified to remove impurities that are not bound to the single-stranded DNA binding protein in the first product to obtain the second product.
  • This step can be performed by various protein separation and purification methods accomplish.
  • the purification method may be affinity chromatography, ion exchange chromatography or gel filtration chromatography (molecular sieve) method, etc., which is not limited in this specification.
  • the method for separating and purifying the first product is affinity chromatography.
  • the affinity chromatography purification can use various affinity chromatography methods for separating and purifying proteins, which is not limited in this description.
  • the single-stranded DNA binding protein may also include purification tags for affinity chromatography purification, such as polyhistidine tags, glutathione-S transferase tags, FLAG tags or Strep-tagII Label.
  • purification tags for affinity chromatography purification such as polyhistidine tags, glutathione-S transferase tags, FLAG tags or Strep-tagII Label.
  • the single-stranded DNA binding protein may have a purification tag, and may be purified using magnetic beads corresponding to the purification tag.
  • a single-stranded DNA-binding protein can include a polyhistidine tag, and the corresponding magnetic beads can be nickel-bearing;
  • a single-stranded DNA-binding protein can include a glutathione-S transferase (GST) tag, and the corresponding magnetic beads can be GST fusion protein purification magnetic beads;
  • single-stranded DNA binding protein can also be Strep-tagII tags, and the corresponding magnetic beads can be Strep-tagII protein purification magnetic beads.
  • the single-stranded DNA binding protein can be tagged with FLAG, and can be purified using a FLAG tag fusion protein purification kit.
  • the first product can be incubated (or incubated) with the affinity chromatography purification medium at room temperature for 30 minutes to 1 hour, and passed through different concentration gradient buffer solutions (such as buffer solutions containing different concentrations of imidazole) Or buffer solutions with different pH gradients to elute components that do not bind to the single-stranded DNA-binding protein, and obtain a second product containing a combination of single-stranded DNA and single-stranded DNA-binding protein.
  • concentration gradient buffer solutions such as buffer solutions containing different concentrations of imidazole
  • buffer solutions with different pH gradients to elute components that do not bind to the single-stranded DNA-binding protein
  • the entire purification process does not involve DNA dyes and carcinogenic agents, and the product has good biological safety, which provides great convenience for clinical trials and other applications; moreover, since the final product purification process does not involve amplification or denaturation, it will not be affected by the target sequence. Therefore, the method for purifying single-stranded DNA provided in this manual is applicable to the purification of single-stranded DNA of various lengths and structures, and has a wide range of applications.
  • the reagent materials required for the whole process are conventional protein purification reagents and tools, the system is easy to scale up and the cost is low.
  • diffuse sequence refers to a sequence containing but not limited to a hairpin structure caused by inverted repeats, a high-low GC sequence or a poly structure, etc., which are difficult to amplify by PCR and result in inefficient synthesis.
  • the remaining liquid can be collected and analyzed to determine whether the single-stranded DNA is well adsorbed to the on an affinity adsorption medium.
  • the magnetic bead method is used for affinity chromatography, after incubating the first product with the magnetic beads for 30-60 minutes, the magnetic beads can be collected using a magnetic separation rack, and the supernatant can be obtained for analysis. As an example only, a certain amount of said supernatant can be taken and analyzed by gel electrophoresis.
  • the result shows that there is no single-stranded DNA that needs to be purified in the supernatant, it means that the DNA in the sample is basically completely adsorbed on the affinity adsorption medium. If the result shows that there is still a large amount of single-stranded DNA in the supernatant, it indicates that the single-stranded DNA binding protein has not completely bound the single-stranded DNA in the sample, or the selected affinity chromatography medium is not effective.
  • the second product can be sampled and analyzed, so as to judge the effect of the affinity chromatography purification. For example, a certain amount of the second product can be taken for gel electrophoresis analysis. If the result shows that there is only single-stranded DNA in the second product, it means that the double-stranded DNA impurities have been removed; if the result shows that there are both single-stranded DNA and double-stranded DNA in the second product, it means that the double-stranded DNA impurities have not been completely removed, so The chosen affinity chromatography procedure is not suitable.
  • non-specific adsorption of double-stranded DNA may be due to an excess of ssDNA-binding protein, which can be achieved by reducing the concentration of ssDNA-binding protein used, reducing the incubation time after mixing the sample with ssDNA-binding protein, or Improvements can be made by incubating and purifying the sample with a small amount of single-stranded DNA-binding protein a second time to avoid the situation where double-stranded DNA impurities are not completely removed.
  • the purpose of purifying the second product is to remove unwanted impurities such as single-stranded DNA binding protein, imidazole, salt, etc. in the second product.
  • This purification step can be selected from any one or several of the following methods: phenol chloroform extraction, DNA adsorption spin column recovery, plasmid extraction ion exchange column recovery, isopropanol precipitation, ethanol precipitation and high performance liquid chromatography
  • the supported molecular sieve chromatography is not limited in this specification.
  • the single-stranded DNA binding protein comprises Escherichia coli (Escherichia coli), phi29 phage (Bacillus phage Nf), Salmonella phage Vi II-E1, thermophile HB8 (Thermus thermophilus), Klebsiella The single-stranded DNA binding protein of Phi K02 phage, Yersinia Py54 phage or Synechococcus phage S-CBS2, its core function fragment, or its homolog sequence or its mutant sequence.
  • the term "core functional fragment” herein refers to a fragment in the protein structure that enables the protein to perform its specific function normally.
  • homologous sequences refer to sequences that are derived from the same ancestor but have gradually diverged in part during evolution.
  • “Mutant sequence” refers to the sequence after natural mutation or artificially induced mutation of the wild type sequence.
  • the core functional fragments, homologue sequences or mutant sequences of the single-stranded DNA binding proteins derived from the above-mentioned microorganisms mentioned here still have the ability to bind to single-stranded DNA.
  • the single-stranded DNA binding protein used in this specification can be a protein that has various sources and can bind to single-stranded DNA, such as derived from other microorganisms not mentioned above, derived from artificial synthesis, etc., this specification There is no restriction on this.
  • the single-stranded DNA binding protein is a single-stranded DNA-binding protein mutant of the above-mentioned microorganism; the single-stranded DNA-binding protein mutant includes at least 70% of the amino acid sequence of the single-stranded DNA-binding protein of the above-mentioned microorganism , 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity and has the ability to bind to single-stranded DNA sequence of functions.
  • accession number of the single-stranded DNA binding protein derived from thermophilic bacteria HB8 is WP_011227803.1 (SEQ ID NO: 9) on NCBI; the single-stranded DNA binding protein mutant derived from thermophilic bacteria HB8
  • the amino acid sequence is SEQ ID NO: 2
  • the nucleotide sequence is SEQ ID NO: 1.
  • the single-stranded DNA binding protein derived from Escherichia coli K-12 has an accession number of AKK14256.2 (SEQ ID NO: 10) on NCBI; the single-stranded DNA binding protein derived from phi29 phage is in The accession number on NCBI is YP_009910721.1 (SEQ ID NO: 11).
  • the single-stranded DNA binding protein comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 2, 9, 10 or 11 %, 96%, 97%, 98%, 99% sequence identity and a sequence that has the function of binding to single-stranded DNA.
  • the single-stranded DNA binding protein comprises the amino acid sequence shown in SEQ ID NO: 2, 9, 10 or 11. In a specific embodiment, the amino acid sequence of the single-stranded DNA binding protein is shown in SEQ ID NO: 2, 9, 10 or 11.
  • the identity of the single-stranded DNA binding protein with the amino acid sequence shown in SEQ ID NO: 2 is greater than or equal to 80%. In some embodiments, the single-stranded DNA binding protein has greater than or equal to 85% identity with the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the single-stranded DNA binding protein has greater than or equal to 90% identity with the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the single-stranded DNA binding protein has greater than or equal to 92% identity with the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the single-stranded DNA binding protein has greater than or equal to 95% identity with the amino acid sequence shown in SEQ ID NO:2.
  • the single-stranded DNA binding protein has greater than or equal to 97% identity with the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the single-stranded DNA binding protein has greater than or equal to 99% identity with the amino acid sequence shown in SEQ ID NO:2. In one embodiment, the amino acid sequence of the single-stranded DNA binding protein is shown in SEQ ID NO:2.
  • sequence identity refers to the amount, typically expressed as a percentage, of the degree of identity between two nucleotide/amino acid sequences (eg, a query sequence and a reference sequence). Typically, prior to calculating the percent identity between two nucleotide/amino acid sequences, an alignment is performed and gaps, if any, are introduced. If at a certain alignment position, the bases or amino acids in the two sequences are the same, the two sequences are considered to be identical or match at this position; the bases/amino acids in the two sequences are different, then they are considered to be inconsistent or mismatched at this position.
  • sequence identity is obtained by dividing the number of matching positions by the total number of positions in the alignment window. In other algorithms, the number of gaps and/or gap lengths are also taken into account.
  • the publicly available alignment software BLAST available at ncbi.nlm.nih.gov
  • BLAST can be used to obtain an optimal sequence alignment and calculate the two nucleotide/amino acid Sequence identity between sequences.
  • PCR amplification and DNA sequencing can be performed on the target product to verify whether the target product contains the complete sequence of the single-stranded DNA. If the sequencing result is consistent with the sequence of the single-stranded DNA, it means that the single-stranded DNA in the target product is complete and can be used in the next step, including but not limited to CRISPR/Cas9 technology to achieve specific gene knockout functions such as delete, knock-in, and fixed-point modification. If the sequencing result is inconsistent with the sequence of the single-stranded DNA, it indicates that the sample may contain multiple single-stranded DNAs or other impurities may be mixed in the purification process, and further purification is required.
  • kits for purifying single-stranded DNA may include the aforementioned single-stranded DNA binding protein and protein purification reagents.
  • the protein purification reagent is an affinity chromatography purification reagent.
  • affinity chromatography purification reagents may include magnetic beads, elution buffer, wash buffer, binding buffer.
  • the binding buffer may contain 50 mM NaH 2 PO 4 , 300 mM NaCl, and have a pH of 8.0.
  • the wash buffer may contain 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, and have a pH of 8.0.
  • the elution buffer may contain 50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, and have a pH of 8.0.
  • the kit can also include one or more nucleic acid primers and one or more nucleic acid probes for PCR amplification and sequencing, thereby verifying whether the target product contains the single-stranded DNA the complete sequence of .
  • the use of a single-stranded DNA-binding protein in purifying single-stranded DNA includes mixing a sample containing single-stranded DNA with a single-stranded DNA binding protein and incubating for a first time to obtain a first product containing the single-stranded DNA and the A conjugate of the single-stranded DNA-binding protein; performing affinity chromatography purification on the first product to obtain a second product; purifying the second product to at least remove the single-stranded DNA-binding protein to obtain a product containing Target product of purified single-stranded DNA.
  • the possible beneficial effects of the method for purifying single-stranded DNA proposed in this specification include but are not limited to: (1) suitable for large-scale purification of single-stranded DNA with high yield; (2) compared with traditional methods (such as gel purification) Compared, the method for purifying single-stranded DNA proposed in this manual adopts an affinity chromatography process, which is fast and efficient; (3) the whole purification process does not involve DNA dyes and carcinogenic agents, and the biological safety of the product is better, which is clinically applicable.
  • the single-stranded DNA method can be applied to the purification of single-stranded DNA of various lengths and structures, and has a wide range of applications; (5)
  • the reagent materials required for the entire process are conventional protein purification reagents and tools, and the system is easy to scale up and low cost. lower.
  • test materials used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified. Quantitative experiments in the following examples, unless otherwise specified, were repeated experiments three times, and the results were averaged.
  • mutant sequence (SEQ ID NO: 1) derived from the SSB gene of thermophile HB8 selected in this manual was synthesized by Nanjing GenScript Biotechnology Co., Ltd.;
  • the pET28a kanamycin-resistant vector was selected as the expression vector, and the SSB gene (SEQ ID NO: 1) was inserted between the restriction site NdeI and HindIII between the 5130 base and the 5923 base to obtain the N-terminal with
  • the polyhistidine-tagged protein expression vector was obtained by homologous recombination as a recombinant plasmid, as shown in Figure 1.
  • the amino acid sequence of the SSB protein encoded by the SSB gene mutant sequence is shown in SEQ ID NO: 2.
  • Escherichia coli BL21 (DE3) strain was transformed with the recombinant plasmid. Single colonies were inoculated into TB large-scale medium containing kanamycin resistance and grown at 37 °C. When the OD 600 reached about 1.2, the cultured cells were induced to express the target protein with 0.5mM IPTG at 15°C/16h. Cells were harvested by centrifugation.
  • the cell pellet was resuspended with lysis buffer (lysis buffer: 50mM Tris, 150mM NaCL, 5% glycerol), then sonicated, and the supernatant was collected after centrifugation. The supernatant was further purified using a nickel-containing affinity chromatography purification medium column to obtain the target protein. After the target protein is filtered and sterilized with a 0.22 ⁇ m filter membrane, it is stored in aliquots.
  • lysis buffer 50mM Tris, 150mM NaCL, 5% glycerol
  • the purified single-stranded DNA binding protein concentration was 2.62mg/ml.
  • Protein purity and molecular weight were determined by reducing SDS-PAGE and Western blot (the main antibody involved was mouse anti-His antibody, purchased from Nanjing GenScript, article number: A00186), as shown in Figure 2, in which the lane M1 is the protein Marker ( Bio-rad, Cat.No.1610374S); lane M2 is protein marker (Genscript, Cat.No.M00521); lane BSA is standard; lane R is purified target single-stranded DNA binding protein, purified single-stranded DNA The band of the binding protein is consistent with the theoretical molecular weight (32.05KDa), and the purity is 90%.
  • This example aims to perform downstream purification of the crude single-stranded DNA product prepared by various methods, so the preparation method of single-stranded DNA will not be repeated here.
  • the rolling circle replication (RCA) method of isothermal amplification of single-stranded DNA was used to prepare single-stranded DNA, and the target sequence 1 was obtained: a crude product of single-stranded DNA (SEQ ID NO: 3) with a length of 1630nt, wherein Contains more non-specific amplification products, and the crude single-stranded DNA product is analyzed by agarose gel electrophoresis, as shown in Figure 3, where lane 1 is the crude product of single-stranded DNA, and the proportion of single-stranded DNA in grayscale analysis is 60.25 %.
  • Step I Binding of ssDNA-binding protein to crude ssDNA
  • Example 2 Dilute the purified single-stranded DNA-binding protein obtained in Example 1 by 5 times, take the diluted single-stranded DNA-binding protein and add it to 10 mL of crude single-stranded DNA product, and mix well.
  • the volume ratio of the mixture is shown in Table 1, Under the condition of 37°C, incubate for 2 hours to ensure that the single-stranded DNA binding protein and the target single-stranded DNA in the crude product can be fully combined to obtain the incubated product.
  • Ni-Charged MagBeads Ni-filled magnetic beads, Nanjing GenScript Biotechnology Co., Ltd., Cat. No. L00295
  • magnetic bead purification method to purify the target sequence 1 that binds to the single-stranded DNA binding protein in step I, and obtain the purified product by affinity chromatography ,Specific steps are as follows:
  • step I Add an equal volume of the incubation product obtained in step I into the test tube, and gently invert the test tube to mix.
  • step 12 Perform agarose gel electrophoresis on the supernatant (purified product) collected in step 11), and compare and analyze the bands before and after purification to preliminarily judge whether the purification is complete.
  • Step III Removal and purification of polyhistidine-tagged ssDNA-binding proteins
  • the affinity chromatography purification product obtained in step II contains by-products such as single-stranded DNA binding protein, imidazole, salt, and this example is carried out in the reaction system by Qiagen-DNA plasmid extraction kit (QIAGEN, Cat.No./ID: 12143) By-products such as protein, imidazole and salt were removed, and the purified product was finally obtained, as shown in Figure 3, in which lane 1 is the crude product of single-stranded DNA, and the proportion of single-stranded DNA in grayscale analysis is 60.25%, and lane 2 is the purification of single-stranded DNA For the product, the proportion of single-stranded DNA in the grayscale analysis was 100%, indicating that the purification product of the target sequence 1 single-stranded DNA with a purity of 100% was obtained through step III.
  • Qiagen-DNA plasmid extraction kit Qiagen-DNA plasmid extraction kit (QIAGEN, Cat.No./ID: 12143)
  • proteases and salts in the reaction system can also be removed by phenol-chloroform extraction, DNA adsorption spin column recovery, isopropanol/ethanol precipitation, or molecular sieve chromatography supported by high performance liquid chromatography.
  • the names and sequences of the amplification primers are as follows:
  • M13R 48: AGCGGATAACAATTTCACACAGGA (SEQ ID NO: 7)
  • the rolling circle replication (RCA) method of isothermally amplifying single-stranded DNA was used to prepare single-stranded DNA, and the target sequence 2 was obtained: a crude product of single-stranded DNA (SEQ ID NO: 8) with a length of 4000nt, which contained More non-specific amplification products.
  • the crude product of single-stranded DNA was analyzed by agarose gel electrophoresis, as shown in Figure 5.
  • Lane 1 is the crude product of single-stranded DNA, and the proportion of single-stranded DNA in grayscale analysis is 53.45%.
  • Step I Binding of ssDNA-binding protein to crude ssDNA
  • Example 2 Dilute the purified single-stranded DNA-binding protein obtained in Example 1 by 5 times, take the diluted single-stranded DNA-binding protein and add it to 10 mL of crude single-stranded DNA product, and mix well.
  • the volume ratio of the mixture is shown in Table 1, Under the condition of 37°C, incubate for 2 hours to ensure that the single-stranded DNA binding protein and the target single-stranded DNA in the crude product can be fully combined to obtain the incubated product.
  • Ni-Charged MagBeads (Nanjing GenScript Biotechnology Co., Ltd., product number L00295) magnetic bead purification method to purify the target sequence 2 that binds to the single-stranded DNA binding protein in step I, and the specific steps are as follows:
  • step I Add an equal volume of the incubation product obtained in step I into the test tube, and gently invert the test tube to mix.
  • test tube Put the test tube on the magnetic separation rack for a few minutes to collect the magnetic beads, and transfer the supernatant containing the eluted protein to a clean test tube.
  • step 12 Perform agarose gel electrophoresis on the supernatant (purified product) collected in step 11), and compare and analyze the bands before and after purification to preliminarily judge whether the purification is complete.
  • Step III Removal and purification of polyhistidine-tagged ssDNA-binding proteins
  • proteases and salts in the reaction system can also be removed by phenol-chloroform extraction, DNA adsorption spin column recovery, isopropanol/ethanol precipitation, or molecular sieve chromatography supported by high performance liquid chromatography.
  • the names and sequences of the amplification primers are as follows:
  • p4000-F GGTATCAGCTCACTCAAAGG (SEQ ID NO: 12)
  • p4000-R CTGTCTCTTGATCAGATCCG (SEQ ID NO: 13)
  • M13R 48: AGCGGATAACAATTTCACACAGGA (SEQ ID NO: 7)
  • p4000-seq9 CCTAACTCCGCCCAGTTCCG (SEQ ID NO: 17)
  • Embodiment 4- use traditional gel electrophoresis and use the comparison of purification method provided by this manual
  • this manual compares the results of the purification method using traditional agarose gel electrophoresis recovery and the purification method using single-stranded DNA binding protein proposed in this manual. Specific steps are as follows:
  • the traditional agarose gel electrophoresis recovery is purified using the AxyPrep DNA Gel Recovery Kit (Axygen, Cat.No.AP-GX-250), and the specific steps are as follows:

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Abstract

提供了一种纯化单链DNA的方法。所述方法可以包括将含有单链DNA的样本与单链DNA结合蛋白混合并孵育第一时间，得到第一产物，所述第一产物中含有所述单链DNA与所述单链DNA结合蛋白的结合物；对所述第一产物进行亲和层析纯化，得到第二产物；对所述第二产物进行纯化以至少去除所述单链DNA结合蛋白，得到含有纯化后的单链DNA的目标产物。还提供了一种用于纯化单链DNA的试剂盒，包括单链DNA结合蛋白和纯化试剂；及单链DNA结合蛋白在纯化单链DNA中的用途。

Description

一种纯化单链DNA的方法
相关申请的交叉引用
本申请要求于2021年10月26日提交的申请号为202111247093.4的中国专利申请的优先权，其内容通过引用被包含于此。
技术领域
本申请涉及生物技术领域，尤其涉及一种纯化单链DNA的方法。
背景技术
CRISPR/Cas9技术是由单链gRNA(Guide RNA)介导的Cas9核酸酶对靶基因的DNA序列进行定向改造的技术，能够高效实现特异性的基因敲除、敲入及定点修饰等功能。单链DNA作为HDR(Homology directed repair，同源介导的双链DNA修复)模板在CRISPR/Cas9基因敲入(Knock-in)实验中具有诸多优势，例如提高编辑效率、编辑准确性，降低脱靶效应和细胞毒性。因此单链DNA模板非常适合应用于基于CRISPR技术的基因的插入、置换和修正，是原代细胞、干细胞基因编辑、基因改造动物模型建立的理想技术方案。
目前有一些能够制备单链DNA样品的方法，例如，WO2020135651公开了将尿嘧啶特异性减除酶(USER)介导的双链DNA自动成环方法和II型限制性内切酶精准切割、等温扩增单链DNA的滚环复制(RCA)巧妙结合起来，从而制备单链DNA样品，该方法也将单链DNA样品的制备产量从常规的几ug提高到几十ug。再例如，Veneziano R等人公开了通过不对称PCR法制备单链DNA模板样品的方式(参见Veneziano R,Shepherd T R,Ratanalert S,et al.In vitro synthesis of gene-length single-stranded DNA[J].Scientific reports,2018,8(1):1-7.)。但随着需求量不断的增大，这些技术仍然存在明显的弊端，例如反应过程中非特异性扩增产物的出现，往往需要凝胶回收的方法进行纯化，大大增加下游纯化工艺的时间和人工成本，阻碍了大规模制备的进程。因此，需要一种能够大规模高效纯化单链DNA的方法。
发明内容
根据本说明书的实施例之一提供了一种纯化单链DNA的方法。所述方法包括：将含有单链DNA的样本与单链DNA结合蛋白混合并孵育第一时间，得到第一产物，所述第一产物中含有所述单链DNA与所述单链DNA结合蛋白的结合物；对所述第一产物进行纯化，得到含有纯化后的单链DNA的目标产物。
在一些实施例中，所述样本与所述单链DNA结合蛋白混合时，所述单链DNA结合蛋白的终浓度为0.5ng/μL-500ng/μL。
在一些实施例中，所述样本与所述单链DNA结合蛋白混合时，所述单链DNA结合蛋白的终浓度为1ng/μL-200ng/μL。
在一些实施例中，所述第一时间为30分钟至12小时。
在一些实施例中，所述第一时间为2-4小时。
在一些实施例中，所述样本与所述单链DNA结合蛋白混合后，在30-37℃下孵育。
在一些实施例中，对所述第一产物进行纯化的步骤包括：对所述第一产物进行分 离纯化，得到第二产物；对所述第二产物进行纯化，以至少去除所述单链DNA结合蛋白，得到含有纯化后的单链DNA的所述目标产物。
在一些实施例中，所述对所述第一产物进行分离纯化的方法为亲和层析法、离子交换层析法或凝胶过滤色谱法。
在一些实施例中，所述对所述第一产物进行分离纯化的方法为亲和层析法。
在一些实施例中，所述单链DNA结合蛋白带有纯化标签。
在一些实施例中，所述纯化标签为多组氨酸标签、谷胱甘肽-S转移酶标签、FLAG标签或Strep-tagII标签。
在一些实施例中，所述对所述第二产物进行纯化的方法选自以下方法中的至少一种：酚氯仿抽提法、DNA吸附离心柱回收法、质粒抽提离子交换柱回收法、异丙醇沉淀法、乙醇沉淀以及分子筛色谱法。
在一些实施例中，所述方法还包括对所述目标产物进行PCR扩增以及DNA测序，以验证所述目标产物中是否含有所述单链DNA的完整序列。
在一些实施例中，所述单链DNA结合蛋白包括来源于大肠杆菌、phi29噬菌体、沙门氏菌噬菌体Vi II-E1、嗜热菌HB8、克雷伯氏菌Phi K02噬菌体、耶尔森式菌Py54噬菌体或聚球藻噬菌体S-CBS2的单链DNA结合蛋白、其核心功能片段、其同源体序列或其突变体序列。
在一些实施例中，所述单链DNA结合蛋白包含与SEQ ID NO：2、9、10或11所示氨基酸序列至少80％序列一致性并具有与单链DNA结合的功能的序列。
本说明书的实施例之一提供了一种用于纯化单链DNA的试剂盒，包括单链DNA结合蛋白和纯化试剂。
在一些实施例中，所述纯化试剂为亲和层析纯化试剂。
本说明书的实施例之一提供了单链DNA结合蛋白在纯化单链DNA中的用途。
附图说明
本说明书将以示例性实施例的方式进一步说明，这些示例性实施例将通过附图进行详细描述。这些实施例并非限制性的，其中：
图1为根据本说明书一些实施例所示的重组表达载体结构示意图；
图2为根据本说明书一些实施例所示的单链DNA结合蛋白还原条件下的SDS-PAGE和Western Blot分析图；
图3为根据本说明书一些实施例所示的目的序列1单链DNA纯化前后对比琼脂糖凝胶电泳图；
图4为根据本说明书一些实施例所示的目的序列1的Sanger测序验证结果示意图；
图5为根据本说明书一些实施例所示的目的序列2单链DNA纯化前后对比琼脂糖凝胶电泳图；
图6为根据本说明书一些实施例所示的目的序列2的Sanger测序验证结果示意图。
具体实施方式
为了更清楚地说明本说明书实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单的介绍。显而易见地，下面描述中的附图仅仅是本说明书的一些示例或实施例，对于本领域的普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图将本说明书应用于其它类似情景。除非从语言环境中显而易见或另做说明，图中相同标号代表相同结构或操作。
应当理解，本文使用的“装置”、“单元”是用于区分不同级别的不同组件、元件或部分的一种方法。然而，如果其他词语可实现相同的目的，则可通过其他表达来替换该词语。
如本说明书和权利要求书中所示，除非上下文明确提示例外情形，“一”、“一个”、“一种”和/或“该”等词并非特指单数，也可包括复数。一般说来，术语“包括”与“包含”仅提示包括已明确标识的步骤和元素，而这些步骤和元素不构成一个排它性的罗列，方法也可能包含其它的步骤或元素。
单链DNA结合蛋白(Single-Stranded DNA binding protein，SSB)是一类能够与单链DNA结合的蛋白，其主要作用是在DNA复制过程中稳定DNA解开的单链，阻止复性和保护单链部分不被核酸酶降解。本说明书提出，可以利用单链DNA结合蛋白对单链DNA的结合能力，对单链DNA进行大规模且高效地纯化，得到较高产量。
根据本说明书的一方面，提供了一种纯化单链DNA的方法。
在一些实施例中，所述方法包括将含有单链DNA的样本与单链DNA结合蛋白混合并孵育第一时间，得到第一产物，所述第一产物中含有所述单链DNA与所述单链DNA结合蛋白的结合物；对所述第一产物进行纯化，得到含有纯化后的单链DNA的目标产物。
在一些实施例中，所述样本可以是大规模生产单链DNA的工艺所得到的产物，因此样本中可能含有非特异性扩增产物，例如双链DNA。为了去除双链DNA杂质，传统方法是使用凝胶电泳法来回收样本中的单链DNA，但这种方法的产量不高，且大大增加下游纯化工艺的时间和人工成本，阻碍了大规模制备的进程。采用本说明书提出的基于单链DNA结合蛋白的纯化方法，可以利用单链DNA结合蛋白对单链DNA的结合能力，对结合单链DNA的单链DNA结合蛋白进行大规模且高效地纯化。
在一些实施例中，所述样本可以指含有一定纯度的单链DNA的任何样本。单链DNA在所述样本中的比例可以是大约10％、20％、30％、40％、50％、60％、70％、80％、90％等，本说明书对此不作限制。其中，单链DNA又称为目的序列。
在一些实施例中，所述样本与所述单链DNA结合蛋白混合时，所述单链DNA结合蛋白的终浓度可以是0.1ng/μL-1000ng/μL。在一些实施例中，所述样本与所述单链DNA结合蛋白混合时，所述单链DNA结合蛋白的终浓度可以是0.5ng/μL-500ng/μL。在一些实施例时，所述样本与所述单链DNA结合蛋白混合时，所述单链DNA结合蛋白的终浓度可以是1ng/μL-200ng/μL。
在一些实施例中，所述样本与所述单链DNA结合蛋白混合后孵育的所述第一时间可以是20分钟至24小时。在一些实施例中，所述样本与所述单链DNA结合蛋白混合后孵育的所述第一时间可以是30分钟至15小时。在一些实施例中，所述样本与所述单 链DNA结合蛋白混合后孵育的所述第一时间可以是30分钟至12小时，例如可以是2小时或4小时。在一些实施例中，所述样本与所述单链DNA结合蛋白混合后孵育的所述第一时间可以是30分钟至6小时。在一些实施例中，所述样本与所述单链DNA结合蛋白混合后孵育的所述第一时间可以是2-4小时。
在一些实施例中，所述样本与所述单链DNA结合蛋白混合后，在30-37℃下孵育。仅作为示例，孵育的温度可以是30℃、32℃、35℃、37℃等。
在一些实施例中，所述对所述第一产物进行纯化的步骤可以包括对所述第一产物进行分离纯化，得到第二产物；对所述第二产物进行纯化，以至少去除所述单链DNA结合蛋白，得到含有纯化后的单链DNA的所述目标产物。
在一些实施例中，对所述第一产物进行分离纯化，以去除第一产物中未与单链DNA结合蛋白结合的杂质，得到第二产物，这一步骤可以通过各种蛋白质的分离纯化方法实现。例如，所述纯化方法可以为亲和层析法、离子交换层析法或凝胶过滤色谱(分子筛)法等，本说明书对此不作限制。在一些实施例中，所述对所述第一产物进行分离纯化的方法为亲和层析法。在一些实施例中，所述亲和层析纯化可以采用各种用于分离纯化蛋白质的亲和层析法，本说明书对此不做限制。
在一些实施例中，所述单链DNA结合蛋白还可以包括用于亲和层析纯化的纯化标签，如多组氨酸标签、谷胱甘肽-S转移酶标签、FLAG标签或Strep-tagII标签。
仅作为示例，所述单链DNA结合蛋白可以带有纯化标签，并且可以采用与纯化标签对应的磁珠进行纯化。例如，单链DNA结合蛋白可以包括多组氨酸标签，对应的磁珠可以带有镍；单链DNA结合蛋白可以包括谷胱甘肽-S转移酶(GST)标签，对应的磁珠可以是GST融合蛋白纯化磁珠；单链DNA结合蛋白还可以是Strep-tagII标签，对应的磁珠可以是Strep-tagII蛋白纯化磁珠。再例如，单链DNA结合蛋白可以采用FLAG标签，可以使用FLAG标签融合蛋白纯化试剂盒进行纯化。
在一些实施例中，可以将第一产物与亲和层析纯化介质在常温下孵育(或温育)30分钟至1小时，通过不同浓度梯度的缓冲液(例如含有不同浓度咪唑的缓冲液)或者不同pH梯度的缓冲液，洗脱不与单链DNA结合蛋白结合的成分，获得含有单链DNA与单链DNA结合蛋白的结合物的第二产物。该方法适用于大规模纯化单链DNA，产量高，速度快，且成本更低(可参见实施例4)。整个纯化过程不涉及DNA染料和致癌制剂，产物的生物安全性较好，为临床试验及其他应用提供了极大便利；并且，由于终产物纯化过程不涉及扩增或变性，不会因为目的序列中含有高难度序列而造成序列错误、丢失或突变，因此本说明书提供的纯化单链DNA的方法可适用于各种长度、各种结构的单链DNA的纯化，应用面广。整个过程所需的试剂材料均为常规蛋白纯化试剂与工具，体系放大方便，成本较低。术语“高难度序列”是指序列中含有但不限于反向重复导致的发夹结构、高低GC序列或poly结构等PCR难以扩增而导致的合成低效的序列。
在一些实施例中，在亲和层析纯化的过程中，可选择地，可以在完成亲和吸附步骤后，收集余下的液体，并进行分析，从而判断单链DNA是否很好地被吸附到了亲和吸附介质上。例如，若使用磁珠法进行亲和层析，可以在将第一产物与磁珠孵育30-60分钟后，使用磁分离架收集磁珠，并获取上清液用于分析。仅作为示例，可以取一定所述上清 液，进行凝胶电泳分析。若结果显示上清液中没有需要纯化的单链DNA，则说明样本中的DNA基本完全被吸附到了亲和吸附介质上。若结果显示上清液中还有大量的单链DNA，说明单链DNA结合蛋白未完全结合样本中的单链DNA，或者所选取的亲和层析介质效果不好。在这种情况下，可以检查所使用的单链DNA结合蛋白是否能够正常结合单链DNA、提高所使用的单链DNA结合蛋白浓度、增加将样本与单链DNA结合蛋白混合后的孵育时间或更换亲和层析介质等方式来进行改善，以提高纯化产量。
在一些实施例中，在完成亲和层析纯化步骤后，可选择地，可以对第二产物进行取样分析，从而判断亲和层析纯化的效果。例如，可以取一定量的所述第二产物，进行凝胶电泳分析。若结果显示第二产物中只有单链DNA，说明双链DNA杂质已被去除；若结果显示第二产物中既有单链DNA，也有双链DNA，说明双链DNA杂质未被完全去除，所选用的亲和层析流程不太合适。例如，可能是由于单链DNA结合蛋白过量，导致非特异性吸附了双链DNA，可以通过降低所使用的单链DNA结合蛋白浓度、减少将样本与单链DNA结合蛋白混合后的孵育时间，或者将样本与少量单链DNA结合蛋白进行二次孵育纯化等方式来进行改善，以避免出现双链DNA杂质未被完全去除的情况。
在一些实施例中，对所述第二产物进行纯化是为了去除所述第二产物中的单链DNA结合蛋白、咪唑、盐等不需要的杂质。这一纯化步骤可以选自以下方法中的任意一种或几种：酚氯仿抽提、DNA吸附离心柱回收、质粒抽提离子交换柱回收、异丙醇沉淀、乙醇沉淀以及高效液相色谱法支持的分子筛色谱法，本说明书对此不做限制。
在一些实施例中，所述单链DNA结合蛋白包括来源于大肠杆菌(Escherichia coli)、phi29噬菌体(Bacillus phage Nf)、沙门氏菌噬菌体Vi II-E1、嗜热菌HB8(Thermus thermophilus)、克雷伯氏菌Phi K02噬菌体、耶尔森式菌Py54噬菌体或聚球藻噬菌体S-CBS2的单链DNA结合蛋白、其核心功能片段、或其同源体序列或其突变体序列。此处术语“核心功能片段”是指该蛋白结构中使该蛋白能正常完成其特定功能的片段。“同源体序列”是指来源于同样的祖先但在进化过程中逐渐产生部分差异的序列。“突变体序列”是指野生型的序列发生自然突变或人为诱导突变之后的序列。这里提到的来源于上述微生物的单链DNA结合蛋白的核心功能片段、其同源体序列或突变体序列仍然具有与单链DNA结合的能力。需要说明的是，本说明书所使用的单链DNA结合蛋白可以是具有各种来源并且能与单链DNA结合的蛋白，如来源于前述未提到的其他微生物、来源于人工合成等，本说明书对此不做限制。
在一些实施例中，所述单链DNA结合蛋白为上述微生物的单链DNA结合蛋白突变体；所述单链DNA结合蛋白突变体包括与上述微生物的单链DNA结合蛋白的氨基酸序列至少70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列一致性并具有与单链DNA结合的功能的序列。
仅作为示例，来源于嗜热菌HB8的单链DNA结合蛋白在NCBI上登录号为WP_011227803.1(SEQ ID NO：9)；所述来源于嗜热菌HB8的单链DNA结合蛋白突变体的氨基酸序列为SEQ ID NO：2，核苷酸序列为SEQ ID NO：1。来源于大肠杆菌K-12(Escherichia coli K-12)的单链DNA结合蛋白在NCBI上登录号为AKK14256.2(SEQ ID NO：10)；所述来源于phi29噬菌体的单链DNA结合蛋白在NCBI上登录号为 YP_009910721.1(SEQ ID NO：11)。在一些实施例中，所述单链DNA结合蛋白包含与SEQ ID NO：2、9、10或11所示氨基酸序列至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列一致性并具有与单链DNA结合的功能的序列。在一些具体实施例中，所述单链DNA结合蛋白包含SEQ ID NO：2、9、10或11所示氨基酸序列。在一个具体实施例中，所述单链DNA结合蛋白的氨基酸序列如SEQ ID NO：2、9、10或11所示。
在一些实施例中，所述单链DNA结合蛋白与SEQ ID NO：2所示的氨基酸序列一致性大于或等于80％。在一些实施例中，所述单链DNA结合蛋白与SEQ ID NO：2所示的氨基酸序列一致性大于或等于85％。在一些实施例中，所述单链DNA结合蛋白与SEQ ID NO：2所示的氨基酸序列一致性大于或等于90％。在一些实施例中，所述单链DNA结合蛋白与SEQ ID NO：2所示的氨基酸序列一致性大于或等于92％。在一些实施例中，所述单链DNA结合蛋白与SEQ ID NO：2所示的氨基酸序列一致性大于或等于95％。在一些实施例中，所述单链DNA结合蛋白与SEQ ID NO：2所示的氨基酸序列一致性大于或等于97％。在一些实施例中，所述单链DNA结合蛋白与SEQ ID NO：2所示的氨基酸序列一致性大于或等于99％。在一个实施方案中，所述单链DNA结合蛋白的氨基酸序列如SEQ ID NO:2所示。
如本文所述的，序列一致性(也称为“序列同一性”)指两核苷酸/氨基酸序列(例如查询序列和参照序列)之间一致性程度的量，一般以百分比表示。通常，在计算两核苷酸/氨基酸序列之间的一致性百分比之前，先进行序列比对(alignment)并引入缺口(gap)(如果有的话)。如果在某个比对位置，两序列中的碱基或氨基酸相同，则认为两序列在该位置一致或匹配；两序列中的碱基/氨基酸不同，则认为在该位置不一致或错配。在一些算法中，用匹配位置数除以比对窗口中的位置总数以获得序列一致性。在另一些算法中，还将缺口数量和/或缺口长度考虑在内。出于本说明书的目的，可以采用公开的比对软件BLAST(可在网页ncbi.nlm.nih.gov找到)，通过使用缺省设置来获得最佳序列比对并计算出两核苷酸/氨基酸序列之间的序列一致性。
在一些实施例中，在获取到目标产物之后，可选择地，可以对目标产物进行PCR扩增以及DNA测序，以验证所述目标产物中是否含有所述单链DNA的完整序列。若测序结果与所述单链DNA的序列一致，则说明目标产物中的单链DNA是完整的，可以用于下一步骤，包括但不限于用于CRISPR/Cas9技术，实现特异性的基因敲除、敲入及定点修饰等功能。若测序结果与所述单链DNA的序列不一致，说明样本中可能含有多种单链DNA或者纯化过程中可能混入了其他杂质，需要进一步纯化。
根据本说明书的另一方面，提供了一种用于纯化单链DNA的试剂盒。在一些实施例中，该试剂盒可以包括前述单链DNA结合蛋白和蛋白纯化试剂。在一些实施例中，所述蛋白纯化试剂为亲和层析纯化试剂。例如，亲和层析纯化试剂可以包括磁珠、洗脱缓冲液、洗涤缓冲液、结合缓冲液。仅作为示例，结合缓冲液可以含有50mM NaH 2PO 4、300mM NaCl，且pH值为8.0。又例如，洗涤缓冲液可以含有50mM NaH 2PO 4、300mM NaCl、10mM咪唑，并且pH值为8.0。再例如，洗脱缓冲液可以含有50mM NaH 2PO 4,、300mM NaCl、250mM咪唑，并且pH值为8.0。
在一些实施例中，所述试剂盒还可以包括一种或多种核酸引物和一种或多种核酸探针，用于PCR扩增以及测序，从而验证目标产物中是否含有所述单链DNA的完整序列。
根据本说明书的又一方面，提供了单链DNA结合蛋白在纯化单链DNA中的用途。例如，在一些实施例中，所述用途包括将含有单链DNA的样本与单链DNA结合蛋白混合并孵育第一时间，得到第一产物，所述第一产物中含有所述单链DNA与所述单链DNA结合蛋白的结合物；对所述第一产物进行亲和层析纯化，得到第二产物；对所述第二产物进行纯化以至少去除所述单链DNA结合蛋白，得到含有纯化后的单链DNA的目标产物。
本说明书提出的纯化单链DNA的方法，可能带来的有益效果包括但不限于：(1)适用于大规模纯化单链DNA，产量高；(2)和传统方法(如凝胶法纯化)相比，本说明书提出的纯化单链DNA的方法采用亲和层析过程，速度快，效率高；(3)整个纯化过程不涉及DNA染料和致癌制剂，产物的生物安全性较好，为临床试验及其他应用提供了极大便利；(4)由于终产物纯化过程不涉及扩增或变性，不会因为目的序列中含有高难度序列而造成序列错误、丢失或突变，因此本说明书提供的纯化单链DNA的方法可适用于各种长度、各种结构的单链DNA的纯化，应用面广；(5)整个过程所需的试剂材料均为常规蛋白纯化试剂与工具，体系放大方便，成本较低。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂公司购买得到的。以下实施例中的定量试验，如无特殊说明，均设置三次重复实验，结果取平均值。
实施例1-单链DNA结合蛋白的制备
1.基因合成与重组载体构建：
本说明书选取的来源于嗜热菌HB8的SSB基因的突变体序列(SEQ ID NO：1)由南京金斯瑞生物科技有限公司合成；
选用pET28a卡那霉素抗性载体作为表达载体，在限制性酶切位点NdeI和HindIII之间即5130碱基和5923碱基之间插入SSB基因(SEQ ID NO：1)获得N端带有多组氨酸标签的蛋白表达载体，通过同源重组方法得到重组质粒，如图1所示。其中，SSB基因突变体序列编码得到的SSB蛋白的氨基酸序列如SEQ ID NO：2所示。
2.蛋白表达：
用重组质粒转化大肠杆菌BL21(DE3)菌株。单菌落接种到含有卡那霉素抗性的TB大规模培养基中，并在37℃下培养。当OD 600达到约1.2时，用0.5mM IPTG在15℃/16h条件下诱导培养细胞表达目标蛋白。通过离心收获细胞。
3.纯化和分析：
将细胞沉淀用裂解缓冲液(裂解缓冲液：50mM Tris,150mM NaCL,5％甘油)重悬，然后超声处理，离心后收集上清液。上清液使用含镍的亲和层析纯化介质柱进一步纯化获得目标蛋白。将目标蛋白使用0.22μm滤膜过滤除菌后，分装储存。
以BSA为标准的Bradford蛋白质测定法测定蛋白浓度，纯化后的单链DNA结 合蛋白浓度为2.62mg/ml。蛋白质纯度和分子量通过还原性SDS-PAGE和Western blot(其中涉及的主要抗体为鼠抗His抗体，购自南京金斯瑞，货号：A00186)结果确定，见图2，其中泳道M1为蛋白Marker(Bio-rad,Cat.No.1610374S)；泳道M2为蛋白Marker(Genscript,Cat.No.M00521)；泳道BSA为标准品；泳道R为纯化后的目标单链DNA结合蛋白，纯化后单链DNA结合蛋白的条带与理论分子量(分子量为32.05KDa)大小一致，纯度为90％。
上述方法所涉及的基因合成与重组载体构建，蛋白表达和纯化分析均由南京金斯瑞生物科技有限公司基因合成服务和蛋白纯化服务完成(https://www.genscript.com.cn/guaranteed-bacterial-protein-expression-services.html？src＝pullmenu)。
实施例2-目的序列1的纯化
本实施例旨在对用各种方法制备的单链DNA粗产物进行下游纯化，故单链DNA制备方法这里不再赘述。按照专利WO2020135651公开的方法，利用等温扩增单链DNA的滚环复制(RCA)方法制备单链DNA，获得目的序列1：长度为1630nt的单链DNA粗产物(SEQ ID NO：3)，其中包含较多的非特异性扩增产物，通过琼脂糖凝胶电泳分析单链DNA粗产物，如图3所示，其中泳道1为单链DNA粗产物，灰度分析单链DNA的占比为60.25％。
本实施例纯化工艺如下：
步骤I-单链DNA结合蛋白与单链DNA粗产物的结合
将实施例1中获得的纯化后的单链DNA结合蛋白稀释5倍，取稀释后的单链DNA结合蛋白加入到10mL单链DNA粗产物中，混匀，混合物体积比例如表1所示，37℃条件下，温育2h，确保单链DNA结合蛋白和粗产物中的目的单链DNA可以充分结合，获得孵育产物。
表1
试剂 单位(mL)
单链DNA粗产物 10
单链DNA结合蛋白 1
步骤II-亲和层析纯化目的序列
采用Ni-Charged MagBeads(Ni填充磁珠，南京金斯瑞生物科技有限公司，货号L00295)磁珠纯化方法纯化步骤I中与单链DNA结合蛋白结合的目的序列1，获得亲和层析纯化产物，具体步骤如下：
1)通过摇动或涡旋小瓶完全重悬Ni-Charged MagBead。
2)将4mL磁珠转移到干净的试管中。
3)将试管放在磁分离架上以收集磁珠，倒出并丢弃上清液。
4)向试管中加入10mL LE Buffer(50mM NaH 2PO 4,300mM NaCl,pH 8.0)并颠倒试管数次以混合。使用磁分离架收集珠子并丢弃上清液。重复此步骤两次。
5)将等体积步骤I获得的孵育产物加入试管中，轻轻颠倒试管混合。
6)20℃下振荡孵育30-60分钟。
7)使用磁分离架收集磁珠并弃去上清液。如有必要，保留用于分析的上清液。
8)加入10ml洗涤缓冲液Wash Buffer(50mM NaH 2PO 4,300mM NaCl,10mM imidazole(咪唑),pH 8.0)至试管中，轻轻颠倒试管混合。将试管放在磁分离架上以收集磁珠，倒出并丢弃上清液。重复洗涤步骤两次。
9)向试管中加入1mL洗脱缓冲液Elution Buffer(50mM NaH 2PO 4,300mM NaCl,250mM imidazole,pH 8.0)，混匀，20℃下振荡孵育5分钟。
10)将试管放在磁分离架上收集磁珠，转移含有洗脱蛋白的上清液至干净的试管中。
11)若有必要重复步骤9)和10)两次。作为后续纯化的样品。
12)将步骤11)中收集到的上清液(纯化产物)进行琼脂糖凝胶电泳，通过纯化前后的条带比对分析，初步判断是否纯化完全。
步骤III-多组氨酸标签的单链DNA结合蛋白的去除和纯化
步骤II中获得亲和层析纯化产物包含单链DNA结合蛋白，咪唑，盐等副产物，本实例通过Qiagen-DNA质粒抽试剂盒(QIAGEN，Cat.No./ID:12143)进行反应体系中蛋白、咪唑和盐等副产物的去除，最终获得纯化产物，见图3，其中泳道1为单链DNA粗产物，灰度分析单链DNA的占比为60.25％，泳道2为单链DNA纯化产物，灰度分析单链DNA占比为100％，说明通过步骤III得到了纯度100％的目的序列1单链DNA纯化产物。
此步骤也可通过酚氯仿抽提、DNA吸附离心柱回收、异丙醇/乙醇沉淀，或高效液相色谱法支持的分子筛色谱法进行反应体系中蛋白酶和盐分的去除。
步骤IV-Sanger测序验证
将步骤III所获得的单链DNA纯化产物稀释至50ng/μL，以单链DNA纯化产物为模板，引物1630nt-F和引物1630nt-R扩增全长，并平末端连接至通用型载体pUC57中。
扩增引物名称与序列如下：
1630nt-F：GGTTTCCTTGAGTGGCAGGC(SEQ ID NO：4)
1630nt-R：TGGCCATTCCTGAAGCAAGG(SEQ ID NO：5)
测序引物名称与序列如下：
M13F(47)：CGCCAGGGTTTTCCCAGTCACGAC(SEQ ID NO：6)
M13R(48)：AGCGGATAACAATTTCACACAGGA(SEQ ID NO：7)
进行Sanger测序，验证结果见图4。从验证结果可以看出目的单链DNA序列与理论序列相符，没出现错误位点，说明该方法是可用于大规模纯化目的单链DNA的切实可行且高效便捷的方法。
实施例3-目的序列2的纯化
按照专利WO2020135651公开的方法利用等温扩增单链DNA的滚环复制(RCA) 方法制备单链DNA，获得目的序列2：长度为4000nt的单链DNA粗产物(SEQ ID NO：8)，其中包含较多的非特异性扩增产物。琼脂糖凝胶电泳分析单链DNA粗产物，如图5所示。其中泳道1为单链DNA粗产物，灰度分析单链DNA的占比为53.45％。
本实施例纯化工艺如下：
步骤I-单链DNA结合蛋白与单链DNA粗产物的结合
将实施例1中获得的纯化后的单链DNA结合蛋白稀释5倍，取稀释后的单链DNA结合蛋白加入到10mL单链DNA粗产物中，混匀，混合物体积比例如表1所示，37℃条件下，温育2h，确保单链DNA结合蛋白和粗产物中的目的单链DNA可以充分结合，获得孵育产物。
步骤II-亲和层析纯化目的序列
采用Ni-Charged MagBeads(南京金斯瑞生物科技有限公司，货号L00295)磁珠纯化方法纯化步骤I中与单链DNA结合蛋白结合的目的序列2，具体步骤如下：
1)通过摇动或涡旋小瓶完全重悬Ni-Charged MagBead。
2)将4mL磁珠转移到干净的试管中。
3)将试管放在磁分离架上以收集磁珠，倒出并丢弃上清液。
4)向试管中加入10mL LE Buffer(50mM NaH2PO4,300mM NaCl,pH 8.0)并颠倒试管数次以混合，使用磁分离架收集珠子并丢弃上清液。重复此步骤两次。
5)将等体积步骤I获得的孵育产物加入试管中，轻轻颠倒试管混合。
6)20℃下振荡孵育30-60分钟。
7)使用磁分离架收集磁珠并弃去上清液。如有必要，保留用于分析的上清液。
8)加入10ml洗涤缓冲液Wash Buffer(50mM NaH2PO4,300mM NaCl,10mM imidazole,pH 8.0)至试管中，轻轻颠倒试管混合。将试管放在磁分离架上数分钟以收集磁珠，倒出并丢弃上清液。重复洗涤步骤两次。
9)向试管中加入1mL洗脱缓冲液Elution Buffer(50mM NaH2PO4,300mM NaCl,250mM imidazole,pH 8.0)，混匀，20℃下振荡孵育5分钟。
10)将试管放在磁分离架上数分钟收集磁珠，转移含有洗脱蛋白的上清液至干净的试管中。
11)若有必要重复步骤9)和10)两次，作为后续纯化的样品。
12)将步骤11)中收集到的上清液(纯化产物)进行琼脂糖凝胶电泳，通过纯化前后的条带比对分析，初步判断是否纯化完全。
步骤III-多组氨酸标签的单链DNA结合蛋白的去除和纯化
亲和层析纯化产物中有加入的单链DNA结合蛋白、咪唑、盐等产物，本实施例通过Qiagen-DNA质粒抽提柱进行反应体系中蛋白酶和盐分的去除。最终获得目的序列2单链DNA纯化产物，见图5，其中泳道1为单链DNA粗产物，灰度分析单链DNA的占比为53.45％，泳道2为单链DNA纯化产物，灰度分析单链DNA占比为100％，说明通过步骤III得到了纯度100％的目的序列2单链DNA纯化产物。
此步骤也可通过酚氯仿抽提、DNA吸附离心柱回收、异丙醇/乙醇沉淀，或高效液相色谱法支持的分子筛色谱法进行反应体系中蛋白酶和盐分的去除。
步骤IV-Sanger测序验证
将步骤III所获得的目的序列2单链DNA纯化产物稀释至50ng/μL，以目的序列2单链DNA纯化产物为模板，引物P-4000-F和引物P-4000-seq4扩增序列1-2000bp区域，引物P-4000-seq11和引物P-4000-R扩增序列1788-4000bp区域，并分别平端连接至通用型载体pUC57中。
扩增引物名称与序列如下：
p4000-F：GGTATCAGCTCACTCAAAGG(SEQ ID NO：12)
p4000-R：CTGTCTCTTGATCAGATCCG(SEQ ID NO：13)
p4000-seq4：CACTACTCAGCGACCTCCAA(SEQ ID NO：14)
p4000-seq11：ATTGTCTCATGAGCGGATAC(SEQ ID NO：15)
测序引物名称与序列如下：
M13F(47)：CGCCAGGGTTTTCCCAGTCACGAC(SEQ ID NO：6)
M13R(48)：AGCGGATAACAATTTCACACAGGA(SEQ ID NO：7)
p4000-seq4：CACTACTCAGCGACCTCCAA(SEQ ID NO：14)
p4000-seq5：GGGCAAGAGCAACTCGGTCG(SEQ ID NO：16)
p4000-seq9：CCTAACTCCGCCCAGTTCCG(SEQ ID NO：17)
p4000-seq10：ATAGGGAGACCCAAGCTGGC(SEQ ID NO：18)
进行Sanger测序，验证结果见图6。从验证结果可以看出目的单链DNA序列与理论序列相符，没出现错误位点，说明该方法是可用于大规模纯化目的序列的切实可行且高效便捷的方法。
实施例4-使用传统凝胶电泳和使用本说明书提供的纯化方法的对比
以一次纯化50mL目的序列1的单链DNA粗产物为例，本说明书对比了使用传统琼脂糖凝胶电泳回收的纯化方法与本说明书提出的使用单链DNA结合蛋白的纯化方法的结果。具体步骤如下：
1.传统琼脂糖凝胶电泳
传统琼脂糖凝胶电泳回收采用AxyPrep DNA凝胶回收试剂盒(Axygen，Cat.No.AP-GX-250)进行纯化，具体步骤如下：
步骤I-2.5％琼脂糖凝胶电泳的制备
1)用蒸馏水将制胶工具洗干净，准备好制胶平板，用琼脂将模具边缘封闭，架好梳子；
2)根据要分离的样品(1630nt ssDNA和dsDNA)大小和稀释后体积配制2.5％的琼脂糖凝胶。以1.5ml上样样品需要制备1块琼脂糖凝胶为例，根据上样体积(稀释后约100ml-150ml，需要制备67-100块琼脂糖凝胶。
3)融化后的琼脂溶液摇晃混匀，倒入电泳槽中，等其凝固；
4)室温下凝固30-40min左右，小心的拔出梳子，取出凝固好的凝胶放入电泳槽内，准备上样；
步骤II-单链DNA粗产物凝胶电泳
1)在电泳槽中倒入电泳缓冲液(TAE)；没过胶面1mm左右，去除胶孔内的气泡；
2)样品稀释：为了更好的区分开ssDNA和dsDNA，将50ml粗产物用水稀释至2-3倍。按照19μL样品加入1ul的回收胶染料比例加入回收胶染料，用抢混匀后加入到凝胶孔内。
3)上样结束，接通电源，红色正极，黑色负极，DNA样品有负极往正极运动，加样孔侧为负，150V-220V电压，运行30min-40min；
4)电压结束，关闭电源，凝胶成像仪上观察ssDNA和dsDNA是否完全分离。
步骤III-凝胶纯化
1)切胶(无核酸污染的干净刀片切)：电泳结束后，在紫外灯下切下对应单链DNA片段大小的琼脂糖凝胶，每三块胶可置于一个50ml的离心管中；
2)每个50ml离心管中加入约7.5ml的Buffer DE-A，混合均匀后于75℃加热，间断混合(每2-3min)，直至凝胶块完全熔化(三块胶约30min-1h)。
3)加入3.75ml的Buffer DE-B，混合均匀；
4)吸取上一步骤中的混合液700μL，转移到一个DNA制备管(置于2ml(试剂盒内提供)离心管)中，总计约1000个制备管。12,000×g离心1min。将滤液重新转移入原制备管，12,000×g离心1min。
5)将制备管置回2ml离心管，每个制备管加500μl Buffer W1，12,000×g离心30s，弃滤液。
6)将制备管置回2ml离心管，每个制备管加700μl Buffer W2，12,000×g离心30s，弃滤液。以同样的方法再每个制备管用700μl Buffer W2洗涤一次12,000×g离心1min。
7)将制备管置回2ml离心管中，12,000×g离心1min，静置30min。
8)将制备管置于洁净的1.5ml离心管(试剂盒内提供)中，在制备膜中央加25-30μl去离子水，室温静置5min。12,000×g离心1min洗脱目的单链DNA。
2.使用单链DNA结合蛋白的纯化方法
按照实施例2的步骤进行，按1:5的比例放大试剂加入量，其余参数不变。
以一次纯化50mL目的序列1的单链DNA粗产物为例，此处统计了使用传统琼脂糖凝胶电泳回收的纯化方法与本说明书提出的使用单链DNA结合蛋白的纯化方法在时间周期、成本及目标产物的产量的不同，如表2所示。
表2
  琼脂糖凝胶电泳的纯化方法 本说明书纯化方法
时间周期(h) 19 5
试剂成本(￥) 2500 820.29
产量(μg) 260 965
结果表明，本说明书纯化方法对比传统琼脂糖凝胶电泳回收的纯化方法在时间周期上缩短了73.7％；所使用的试剂成本减少了67.2％，而目标产物的产量增加了2.7倍。
本领域的技术人员应当理解，以上实施例仅为说明本说明书，而不对本说明书构成限制。凡在本说明书的精神和原则内所作的任何修改、等同替换和变动等，均应包含在本说明书的保护范围之内。

Claims (18)

  1. 一种纯化单链DNA的方法，其特征在于，包括：
    将含有单链DNA的样本与单链DNA结合蛋白混合并孵育第一时间，得到第一产物，所述第一产物中含有所述单链DNA与所述单链DNA结合蛋白的结合物；
    对所述第一产物进行纯化，得到含有纯化后的单链DNA的目标产物。
  2. 如权利要求1所述的方法，其特征在于，所述样本与所述单链DNA结合蛋白混合时，所述单链DNA结合蛋白的终浓度为0.5ng/μL-500ng/μL。
  3. 如权利要求1所述的方法，其特征在于，所述样本与所述单链DNA结合蛋白混合时，所述单链DNA结合蛋白的终浓度为1ng/μL-200ng/μL。
  4. 如权利要求1所述的方法，其特征在于，所述第一时间为30分钟至12小时。
  5. 如权利要求1所述的方法，其特征在于，所述第一时间为2-4小时。
  6. 如权利要求1所述的方法，其特征在于，所述样本与所述单链DNA结合蛋白混合后，在30-37℃下孵育。
  7. 如权利要求1所述的方法，其特征在于，对所述第一产物进行纯化的步骤包括：
    对所述第一产物进行分离纯化，得到第二产物；
    对所述第二产物进行纯化，以至少去除所述单链DNA结合蛋白，得到含有纯化后的单链DNA的所述目标产物。
  8. 如权利要求7所述的方法，其特征在于，所述对所述第一产物进行分离纯化的方法为亲和层析法、离子交换层析法或凝胶过滤色谱法。
  9. 如权利要求8所述的方法，其特征在于，所述对所述第一产物进行分离纯化的方法为亲和层析法。
  10. 如权利要求9所述的方法，其特征在于，所述单链DNA结合蛋白带有纯化标签。
  11. 如权利要求10所述的方法，其特征在于，所述纯化标签为多组氨酸标签、谷胱甘肽-S转移酶标签、FLAG标签或Strep-tagII标签。
  12. 如权利要求7所述的方法，其特征在于，所述对所述第二产物进行纯化的方法选自以下方法中的至少一种：酚氯仿抽提法、DNA吸附离心柱回收法、质粒抽提离子交换柱回收法、异丙醇沉淀法、乙醇沉淀以及分子筛色谱法。
  13. 如权利要求1所述的方法，其特征在于，所述方法还包括对所述目标产物进行PCR扩增以及DNA测序，以验证所述目标产物中是否含有所述单链DNA的完整序列。
  14. 如权利要求1所述的方法，其特征在于，所述单链DNA结合蛋白包括来源于大肠杆菌、phi29噬菌体、沙门氏菌噬菌体Vi II-E1、嗜热菌HB8、克雷伯氏菌Phi K02噬菌体、耶尔森式菌Py54噬菌体或聚球藻噬菌体S-CBS2的单链DNA结合蛋白、其核心功能片段、其同源体序列或其突变体序列。
  15. 如权利要求14所述的方法，其特征在于，所述单链DNA结合蛋白包含与SEQ ID NO：2、9、10或11所示氨基酸序列相比具有至少80％序列一致性并具有与单链DNA结合的功能的序列。
  16. 一种用于纯化单链DNA的试剂盒，其特征在于，包括单链DNA结合蛋白和纯化试剂。
  17. 如权利要求16所述的试剂盒，其特征在于，所述纯化试剂为亲和层析纯化试剂。
  18. 单链DNA结合蛋白在纯化单链DNA中的用途。
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